In Vivo Chemical Genomics with Random Cyclized Peptides

We have developed and applied a novel strategy that can best be described as in vivo chemical genomics, a concept where populations of any transformable organism may be screened for consequences of novel RNAs or peptides. We created a library of ~800,000 random DNA sequences biased only by third-position nucleotide substitutions that suppress the frequency of termination codons. The sequences may be shuttled to any plant, microbial, or animal expression vector with recombination cloning. We then generated large populations of Arabidopsis thaliana plants, each expressing a randomized DNA sequence, presumably giving rise to synthetic RNA species and/or the peptides they encode. These novel molecules are produced within the context of the cell and have been shown to affect plant biology with a relatively high frequency, as evidenced by diverse phenotypes. This chapter provides the protocols necessary to construct the libraries and isolate plants expressing randomized DNA sequences.